Expression of proteins in gram-negative bacteria wherein the ratio of periplasmic volume to cytoplasmic volume is between 0.5:1 and 10:1

ABSTRACT

Provided are modified Gram-negative bacteria having an increased periplasmic volume. Also provided are methods of expressing exogenous genes in the bacteria and targeting protein production to the periplasmic space.

CROSS-REFERENCE TO RELATED APPLICATIONS AND APPLICATIONS INCORPORATED BYREFERENCE

This application is a National Phase application of International PatentApplication No. PCT/US17/24857, filed Mar. 29, 2017, entitled“EXPRESSION OF PROTEINS IN GRAM-NEGATIVE BACTERIA WHEREIN THE RATIO OFPERIPLASMIC VOLUME TO CYTOPLASMIC VOLUME IS BETWEEN 0.5:1 AND 10:1”,which claims priority from U.S. Provisional Patent Application62/314,924, filed Mar. 29, 2016, titled MODIFIED BACTERIA AND USESTHEREOF; the disclosures of which are incorporated by reference hereinin their entirety.

BACKGROUND Field

The invention relates to modified Gram-negative bacteria having anincreased periplasmic volume, and methods of expressing exogenous genestherein. The bacteria are useful for targeting recombinant proteinproduction to the periplasmic space.

Description of the Related Art

In spite of longstanding efforts to optimize prokaryotic expressionsystems, a number of obstacles still remain to obtaining sufficientyields of functionally active gene products, including the formation ofinclusion bodies, incorrect folding of the expressed protein, toxicityfor the producer cells and degradation by proteases. A variety ofalternative expression systems are being developed and evaluated toproduce recombinant proteins more effectively.

Escherichia coli is the most commonly used host for the production ofrecombinant proteins. In order to obtain the target exogenous proteinsexpressed intracellularly in recombinant E. coli, however, celldisruption is necessary, which can increase of pyrogen level (mainlyfrom the cell membrane composition), increase sample impurities anddecrease protein activities. Particularly, the formation of inclusionbodies often occurs when the target protein is intracellularlyoverexpressed. To overcome these problems, extracellular secretion ofexogenous proteins in recombinant E. coli is becoming an increasinglypopular choice. In large-scale industrial production of exogenousproteins, the extracellular excretion of target proteins can remove thecell disruption step, offer a better environment for protein folding andreduce the risk of intracellular enzyme degradation (Mergulhao et al.,2005, Biotechnol Adv 23: 177-202). Additionally, the extracellularsecretion of target proteins can improve the recombinant protein yieldbecause the target protein accumulation is not limited in periplasmic orintracellular space (Makrides, 1996, Microbiol Rev 60: 512-538; Fu etal., 2005, Biotechnol Prog 21: 1429-1435).

The primary challenge with the extracellular secretion of recombinantproteins are the difficulties inherent in protein translocation acrossboth the cell membrane and the outer membrane of E. coli cells (Koebniket al., 2000, Mol Microbiol 37: 239-253; Choi and Lee, 2004, ApplMiccrobiol Biotechnol 64: 625-635). While periplasmic expression ofrecombinant proteins can often be achieved with the help of a signalpeptide, the available methods to overcome the outer membrane barrierfor extracellular production of recombinant proteins are much morelimited. In order to solve this problem, various genetic attempts havebeen made to facilitate the extracellular secretion of recombinantproteins in E. coli, including manipulation of transport pathways(Sugamata and Shiba, 2005, Appl Environ Microbiol 71: 656-662),optimization of codon and signal sequence (Takemori et al., 2012,Protein Expr Purif 81: 145-150), fusion expression of carrier proteinwhich can be normally secreted extracellularly (Fernandez et al., 2000,Appl Environ Microbiol 66: 5024-5029; Choi and Lee, 2004) and fusionexpression of outer membrane protein F (Jeong and Lee, 2002, ApplEnviron Microbiol 68: 4979-4985), YebF (Zhang et al., 2006, NatBiotechnol 24: 100-104) or osmotically inducible protein Y (Qian et al.,2008, Biotechnol Bioeng 101: 587-601). In addition, the coexpression oflysis-promoting proteins such as bacteriocin release protein (BRP) (vander Wal et al., 1995, Appl Microbiol Biotechnol 44: 459-465) or colicinE1 lysis protein (Kil) (Robbens et al., 1995, Protein Expr Purif 6″481-486), as well as the use of wall-less strains (the so-calledL-forms) (Gumpert and Hoischen, 1998, Current Opinion in Biotechnology,9: 506-509) have also been reported.

Meanwhile, many fermentation techniques, including changes of culturemedium compositions (Fu, 2010, Appl Microbiol Biotechnol 88: 75-86),temperature (Rinas and Hoffmann, 2004, Biotechnol Prog, 20: 679-687),aeration and calcium ion (Shokri et al., 2003, Appl Microbiol Biotechnol60: 654-664), osmotic pressure and induction conditions (Orr et al.,2012, J Biotechnol 161, 19-26), as well as the addition of supplementssuch as glycine (Yang et al., 1998, Appl Environ Microbiol 64:2869-2874) and Triton X-100 (Fu et al., 2005, Biotechnol Prog 21:1429-1435; Fu, 2010), have been explored to achieve the extracellularproduction of recombinant proteins in E. coli. The main disadvantage ofthe fermentation control for the extracellular production of targetproteins is that the fermentation conditions vary greatly with differenttarget proteins.

Leaky strains (including the E. coli Sec pathway) offer an alternativemeans for transporting periplasmic-directed recombinant proteins intomedia that overcomes the uncertainty of the fermentation conditions.Leaky strains can be constructed by knocking out genes related to thebiosynthesis of cell wall and membrane, especially of the outer membranegenes such as lpp encoding Braun's lipoprotein (Shin and Chen, 2008,Biotechnol Bioeng 101: 1288-1296) of E. coli. More recently, Chen et al.(Microbial Biotechnology, 2014, 7, 360-370) constructed several leakystrains of E. coli JM109 (DE3), including mrcA, mrcB, pal and lpp(single-gene knock-out), and lpp mrcB, mrcA lpp, lpp pal, mrcA pal andmrcB pal (double-gene knock-out), by an inframe deletion method toimprove the extracellular secretory levels of their target proteins.Extracellular yields of recombinant protein Trx-hPTH (human parathyroidhormone 1-84 coupled with thioredoxin as a fusion partner) from themutants with double deletion were significantly higher than those fromthe mutants with single deletion under the same conditions. In addition,mutants with inframe single/double deletion of genes, mrcB and lpp,could not cause the efficient leakage of the target protein due toprotein expression in the cytoplasm rather than the periplasm.Accordingly, while the main advantage of leaky strains is that noadditives are needed to induce extracellular protein production, themain disadvantage is that their secretory selectivity is not high,suggesting that these genes affect the structure of the outer membranebut do not participate in the active transport of target protein(s).

L-form bacteria, or L-forms, are bacterial strains derived from parentspecies (N-forms) that are able to grow as cell wall-deficient(spheroplast type) or as cell wall-less (protoplast type) cells. See,Madoff S (Ed): The Bacterial L-Forms. New York: Marcel Dekker Inc.,1986; Mattmann L H (Ed): Cell Wall Deficient Forms. Boca Raton: CRCPress; 1993; and Gumpert J, Taubeneck U: Characteristic properties andbiological significance of stable protoplast type L-forms. InProtoplasts, Lecture Proceedings of the 6^(th) International ProtoplastSymposium: Basel. Experientia 1983, 46(suppl):227-241.

Protoplast type L-forms have been cultivated in the cell wall-less stateand represent genetically stable mutants showing extreme pleiotropicchanges, including the inability to form cell walls, capsules, flagella,pili, spores and mesosomes, altered colony and cell morphology,qualitative and quantitative changes in the lipid and protein componentsof the cytoplasmic membrane, the absence of extracelluar proteolyticactivities, resistance against bacteriophages and the incapability topropagate outside laboratory conditions. See, Gumpert and Taubeneck(supra); and Hoischen et al., Lipid and fatty acid composition ofcytoplasmic membranes from Streptomyces hygroscopicus and its stableprotoplast type L-form. J Bacteriol 1997, 179:3430-3436.

Gumpert and Hoischen (Current Opinion in Biotechnology, 1998, 9:506-509)describe expression systems in which cell wall-less L-form strains ofProteus mirabilis, Escherichia coli, Bacillus subtilis, and Streptomyceshydroscopicus were used to synthesize various recombinant proteins inconsiderable amounts as soluble, functionally active products. Therecombinant proteins were secreted by the L-form cells into thesurrounding growth medium by an active translocation process thatrequired appropriate signal peptides. Among the proteins synthesizedwere correctly processed antibodies and miniantibodies, indicating thatthe appropriate post-translational modifications (correct folding,formation of disulfide bonds, and dimerization) had occurred. Theauthors noted that because the L-form strains lacked a periplasmiccompartment this is not a necessary prerequisite for post-translationalprocessing and that the cytoplasmic membrane of the L-form cells plays arole in these modification processes. The L-form cells were moresensitive to environmental influences than widely-used E. coliexpression systems and they needed more careful handling, in particular,control of the inoculum, the avoidance of contacts with membrane-activesurfactants and other aggressive substances, and complex growth media.The authors concluded that the most important advantage of their L-formexpression system was the removal of the synthesized protein by activetranslocation through the cytoplasmic membrane and secretion into thesurrounding growth medium, and that it was probably not useful for largescale fermentations.

Accordingly, there is clearly still a need for improved methods for thefermentative preparation of proteins. The recombinant bacteria, cellculture media, and processes described herein help meet these and otherneeds.

BRIEF SUMMARY

The present invention solves the foregoing problems in the prior art byproviding compositions and methods for the enhanced periplasmicproduction of recombinant proteins. In particular, modified bacterialcells are provided exhibiting a novel physiological state which inhibitscell division and promotes the growth of the periplasmic space incomparison to the cytoplasmic space. As demonstrated for the first timeherein, recombinant protein production in these cells is dramaticallyincreased compared with that in non-switched cells. Structurally, thecells comprise both inner and outer membranes but lack a functionalpeptidoglycan cell wall, while the cell shape is spherical and increasesin volume over time. Notably, while the periplasmic space normallycomprises only 10-20% of the total cell volume, the periplasmiccompartment of the switched state described herein can comprise morethan 20%, 30%, 40% or 50% and up to 60%, 70%, 80% or 90% of the totalcell volume. In some cases, this increased periplasmic space providesfor dramatically increased expression of recombinant proteins into theperiplasmic space.

In one aspect, modified bacterial cells are provided exhibiting thisswitched phenotype, where the periplasmic space in the subject bacterialcells comprises at least about 20% or 25% of the total cell volume, morepreferably at least about 30%, 35%, 40%, or 45% of the total cellvolume, still more preferably at least about 50%, 55%, 60%, 65%, or 70%of the total cell volume, and most preferably at least about 75%, 80%,85% or 90% of the total cell volume.

Preferably, the modified bacterial cells of the subject invention arederived from Gram-negative bateria, e.g. selected from:gammaproteobacteria and alphaproteobacteria. In particularly preferredembodiments, the bacterium is selected from: Escherichia coli, Vibrionatriegens, Pseudomonas fluorescens, Caulobacter crescentus,Agrobacterium tumefaciens, and Brevundimonas diminuta. In specificembodiments, the bacterium is Escherichia coli, e.g. strain BL21,BL21(DE3), or K12.

In some embodiments, the modified bacterial cells according to thepresent invention have a ratio of periplasmic volume to cytoplasmicvolume between about 0.5:1 and about 10:1, between about 0.5:1 and about5:1, between about 0.5:1 and about 1:1, between about 1:1 and about10:1, between about 1:1 and about 5:1, or between about 5:1 and about10:1.

In some embodiments, a modified bacterial cell of the present inventionis a coccus having a longest dimension of about 2 μm to about 16 μm,more preferably about 4 μm to about 16 μm, still more preferably about 8μm to about 16 μm, or about 2 μm to about 8 μm, or about 4 μm to about 8μm, or about 2 μm to about 4 μm.

Preferably, the modified bacterial cells of the subject inventionfurther comprise an exogenous gene encoding a protein of interest.Proteins of interest can be therapeutic, e.g., antibodies, hormones,growth factors, vaccines, and any other functional and/or structuralproteins and enzymes of medical interest, as well as non-therapeutic,e.g., collagen and derivatives thereof, albumin, ovalbumin, rennet,fibrin, casein (including αS1, αS2, β, κ), elastin, keratin, myosin,fibronectin, laminin, nidogen-1, vitronectin, silk fibroins, prolylhydroxylases, lysyl hydroxylases, glycosyltransferases, hemeproteins,and any other structural or non-structural proteins or enzymes ofcommercial and/or academic interest. Proteins of interest herein mayexclude fluourescent proteins. In certain embodiments, the protein isother than mCherry or green fluorescent protein.

In one embodiment, the exogenous gene is integrated into the hostbacterial cell genome. In another embodiment, the bacterial cellcomprises an expression vector comprising the exogenous gene. In someembodiments, the expression vector is free of a marker encoding forresistance to an inhibitor of bacterial cell peptiglycan biogenesis. Inan exemplary embodiment, the expression vector comprises a pET plasmid.In a particular embodiment, the expression vector comprises plasmidpET28a.

In one embodiment, expression of the exogenous gene is constitutive. Inanother embodiment, expression of the exogenous gene is inducible. Insome embodiments, expression of the exogenous gene is inducible by aninducer selected from, e.g. isopropyl-β-d-1-thiogalactopyranoside,lactose, arabinose, maltose, tetracycline, anhydrotetracycline,vavlycin, xylose, copper, zinc, and the like.

In one embodiment, the modified bacterial cells further comprise anucleic acid sequence encoding a signal peptide operably linked to theexogenous gene, wherein the signal peptide directs cotranslationalexport of the protein from the cytoplasm to the periplasm. In someembodiments, the signal peptide is derived from a protein component ofthe Sec and Tat secretion pathways. In particular embodiments, thesignal peptide is derived from DsbA, pelB, OmpA, TolB, MalE, lpp, TorA,or HylA. For example, the signal peptide can be an N-terminal portion ofDsbA, pelB, OmpA, TolB, MalE, lpp, TorA, or HylA that directscotranslational export of the protein from the cytoplasm to theperiplasm. In some cases, the signal peptide can contain at least 10%,25%, 50%, 75%, 95%, 99%, or all of a peptide selected from DsbA, pelB,OmpA, TolB, MalE, lpp, TorA, or HylA.

In some embodiments, the modified bacterial cell comprises a coccus formof an ampicillin sensitive (amp^(S)) and/or fosfomycin-sensitivebacillus strain, wherein the coccus form and the bacillus strain aregenetically identical.

In another aspect, cell cultures are provided comprising bacterial cellshaving an enlarged periplasmic space in a culture medium comprising amagnesium salt, wherein the concentration of magnesium ions in themedium is at least about 4, 5 or 6 mM. In further embodiments, theconcentration of magnesium ions in the medium is at least about 7, 8, 9or 10 mM. In some embodiments, the concentration of magnesium ions inthe medium is between about 6 mM and about 20 mM. In some embodiments,the magnesium salt is selected from: magnesium sulfate and magnesiumchloride.

Preferably, the cell culture of the subject invention further comprisesan osmotic stabilizer, including, e.g. sugars (e.g., arabinose, glucose,sucrose, glycerol, sorbitol, mannitol, fructose, galactose, saccharose,maltotrioseerythritol, ribitol, pentaerythritol, arabitol, galactitol,xylitol, iditol, maltotriose, and the like), betaines (e.g.,trimethylglycine), proline, one or more salts such as an ammonium,potassium, or sodium salt (e.g., sodium chloride), one or more polymers(e.g., polyethylene glycol, polyethylene glycol monomethylether,polysucrose, polyvinylpyrrolidone, polypropylene glycol), or acombination thereof. In some cases, the concentration of the osmoticstabilizer(s) in the medium is at least about 4%, 5%, 6%, or 7% (w/v).In further embodiments, the concentration of osmotic stabilizer is atleast about 8%, 9%, or 10% (w/v). In some embodiments, the concentrationof the osmotic stabilizer in the medium is between about 5% to about 20%(w/v).

In some embodiments, the cell culture may further comprise ammoniumchloride, ammonium sulfate, calcium chloride, amino acids, iron(II)sulfate, magnesium sulfate, peptone, potassium phosphate, sodiumchloride, sodium phosphate, and yeast extract. In some embodiments, thecell culture is free of animal-derived components. In some embodiments,the cell culture comprises, consists essentially of, or is in, a definedmedium.

In some embodiments, the cell culture comprises from about 1×10⁸bacterial cells per mL of culture volume to about 1×10¹⁰ bacterial cellsper mL in a volume of at least about 1 L (e.g., from about 1 L to about500,000 L, from about 1 L to about 10,000 L, from about 1 L to about1,000 L, from about about 1 L to about 500 L, or from about 1 L to about250 L). In some embodiments, the cell culture comprises from about 4×10⁸bacterial cells per mL of culture volume to about 1×10⁹ bacterial cellsper mL in a volume of at least about 1 L (e.g., from about 1 L to about500,000 L, from about 1 L to about 10,000 L, from about 1 L to about1,000 L, from about about 1 L to about 500 L, or from about 1 L to about250 L).

In some embodiments, the cell culture comprises at least one exogenousantibiotic inhibitor of bacterial cell peptidoglycan biogenesis. In someembodiments, the cell culture comprises at least two structurallydistinct exogenous antibiotic inhibitors of bacterial cell peptidoglycanbiogenesis. In some cases, the at least two structurally distinctexogenous antibiotic inhibitors of bacterial cell peptidoglycanbiogenesis inhibit different components of a peptidoglycan biogenesispathway in the bacterial cell (e.g., the at least two structurallydistinct exogenous antibiotic inhibitors inhibit different enzymes ofthe peptidoglycan biogenesis pathway in the bacterial cell). In somecases, the cell culture comprises an exogenous antibiotic inhibitor of atransglycosylase component of bacterial cell peptidoglycan biogenesis.In some cases, the cell culture comprises an exogenous antibioticinhibitor of a transpeptidase component of bacterial cell peptidoglycanbiogenesis. In some cases, the cell culture comprises an exogenousantibiotic inhibitor of UDP-N-acetylmuramyl (UDP-MurNAc)-pentapeptidebiogenesis or UDP-N-acetylglucosamine (UDP-GlcNAc) biogenesis.

In some cases, the cell culture comprises an exogenous antibioticinhibitor of MurA, MurB, MurC, MurD, MurE, MurF, MraY, MurG, FemX, FemA,FemB, FtsW, or a penicillin binding protein (PBP). In some cases, thecell culture comprises at least two structurally distinct exogenousantibiotic inhibitors, wherein the at least two structurally distinctexogenous antibiotic inhibitors inhibit different proteins selected fromMurA, MurB, MurC, MurD, MurE, MurF, MraY, MurG, FemX, FemA, FemB, FtsW,or a penicillin binding protein (PBP). In some embodiments, the cellculture comprises at least one or at least two structurally distinctantibiotic(s) selected from: β-lactam antibiotics, phosphonic acidantibiotics, polypeptide antibiotics, D-cycloserine, and glycopeptideantibiotics, wherein the antibiotic(s) inhibit peptidoglycan biogenesisin the bacterial cell.

In some embodiments, the culture medium comprises at least one reactiveoxygen species (ROS) scavenger (e.g., reduced glutathione (GSH), or athiol containing non-peptidic small molecule having a molecular weightfrom about 70 g/mol to about 350 g/mol, or from about 75 g/mol to about155 g/mol).

In some embodiments, the culture medium comprises an antibiotic (e.g.,second or third antibiotic) that selects for the presence of aselectable marker in an expression vector that comprises an exogenousgene encoding for a protein of interest. In some embodiments, theantibiotic that selects for the presence of a selectable marker in theexpression vector is not an inhibitor of peptidoglycan biogenesis in thebacterial cell.

In another aspect, methods of producing an exogenous protein of interestare provided (e.g., using one or more of the foregoing cell culturesand/or bacterial cells), comprising: a) culturing a Gram-negativebacterial cell in a medium comprising a magnesium salt, wherein theconcentration of magnesium ions in the culture medium is at least about4, 5 or 6 mM, and wherein the bacterial cell comprises an exogenous geneencoding the protein of interest; b) inhibiting peptidoglycan biogenesisin the bacterial cell; and c) harvesting the protein from the medium. Infurther embodiments, the concentration of magnesium ions in the mediumis at least about 7, 8, 9 or 10 mM. In some embodiments, theconcentration of magnesium ions in the medium is between about 6 mM andabout 20 mM. In some embodiments, the magnesium salt is selected from:magnesium sulfate and magnesium chloride. In some embodiments, theculturing of a), or a portion thereof and/or the inhibiting of b), or aportion thereof is performed in one or more of the foregoing cellcultures. In some embodiments, expression of the exogenous gene encodingthe protein of interest is induced, e.g., by adding an inducer to themedium, during step b). In some embodiments, expression of the exogenousgene encoding the protein of interest is induced, e.g., by adding aninducer to the medium, before step b). In some embodiments, expressionof the exogenous gene encoding the protein of interest is induced, e.g.,by adding an inducer to the medium, after step b) has been initiated.

Preferably, the cell culture of the subject invention further comprisesan osmotic stabilizer, including, e.g. sugars (e.g., arabinose, glucose,sucrose, glycerol, sorbitol, mannitol, fructose, galactose, saccharose,maltotrioseerythritol, ribitol, pentaerythritol, arabitol, galactitol,xylitol, iditol, maltotriose, and the like), betaines (e.g.,trimethylglycine), proline, one or more salts such as an ammonium,potassium, or sodium salt (e.g., sodium chloride), one or more polymers(e.g., polyethylene glycol, polyethylene glycol monomethylether,polysucrose, polyvinylpyrrolidone, polypropylene glycol), or acombination thereof. In some cases, the concentration of the osmoticstabilizer(s) in the medium is at least about 4%, 5%, 6%, or 7% (w/v).In further embodiments, the concentration of osmotic stabilizer is atleast about 8%, 9%, or 10% (w/v). In some embodiments, the concentrationof the osmotic stabilizer in the medium is between about 5% to about 20%(w/v).

In some embodiments, the cell culture may further comprise ammoniumchloride, ammonium sulfate, calcium chloride, amino acids, iron(II)sulfate, magnesium sulfate, peptone, potassium phosphate, sodiumchloride, sodium phosphate, and yeast extract.

The bacterial cell may be cultured continuously or discontinuously; in abatch process, a fed-batch process or a repeated fed-batch process. Insome embodiments, steps b) and c) occur sequentially. In otherembodiments, steps b) and c) occur simultaneously. In some embodiments,step c) is performed at least 1 hour after step b).

In some embodiments, the inhibiting peptidoglycan biogenesis in thebacterial cell is performed by adding an antibiotic to the medium. Insome cases, the inhibiting peptidoglycan biogenesis in the bacterialcell is performed by adding two or more structurally distinctantibiotics to the medium. In some cases, the antibiotic or antibioticsare selected from: β-lactam antibiotics (e.g. penicllins,cephalosporins, carbapenems, and monobactams), phosphonic acidantibiotics, polypeptide antibiotics, and glycopeptide antibiotics. Inparticular embodiments, the antibiotic or antibiotics are selected fromalafosfalin, amoxicillin, ampicillin, aztreonam, bacitracin,carbenicillin, cefamandole, cefotaxime, cefsulodin, cephalothin,fosmidomycin, methicillin, nafcillin, oxacillin, penicillin g,penicillin v, fosfomycin, primaxin, D-cycloserine, and vancomycin. Insome embodiments, the antibiotic or antibiotics are selected frominhibitors of MurA, MurB, MurC, MurD, MurE, MurF, MraY, MurG, FemX,FemA, FemB, FtsW, or a penicillin binding protein (PBP). In someembodiments, the inhibiting peptidoglycan biogenesis in the bacterialcell is performed by adding an inhibitor of MurA and an inhibitor of apenicillin binding protein (PBP) to the medium. In some embodiments, theinhibiting peptidoglycan biogenesis in the bacterial cell is performedby adding an inhibitor of MurA to the medium. In some embodiments, theinhibiting peptidoglycan biogenesis in the bacterial cell is performedby adding an inhibitor of PBP to the medium.

Preferably, the modified bacterial cells of the subject invention arederived from Gram-negative bateria, e.g. selected from:gammaproteobacteria and alphaproteobacteria. In particularly preferredembodiments, the bacterium is selected from: Escherichia coli, Vibrionatriegens, Pseudomonas fluorescens, Caulobacter crescentus,Agrobacterium tumefaciens, and Brevundimonas diminuta. In specificembodiments, the bacterium is Escherichia coli, e.g. strain BL21(DE3).

In one embodiment, the exogenous gene is integrated into the hostbacterial cell genome. In another embodiment, the bacterial cellcomprises an expression vector comprising the exogenous gene. In someembodiments, the medium comprises an antiobiotic that selects for thepresence of the expression vector. In some embodiments, the antiobioticthat selects for the presence of the expression vector is not aninhibitor of peptidoglycan biogenesis in the bacterial cell. In anexemplary embodiment, the expression vector comprises a pET plasmid. Ina particular embodiment, the expression vector comprises plasmid pET28a.

In a further embodiment, the subject methods comprise inducingexpression of the exogenous gene. In some embodiments, expression of theexogenous gene is inducible by an inducer selected from, e.g.isopropyl-β-d-1-thiogalactopyranoside, lactose, arabinose, maltose,tetracycline, anhydrotetracycline, vavlycin, xylose, copper, zinc, andthe like.

In one embodiment, the modified bacterial cells further comprise anucleic acid sequence encoding a signal peptide operably linked to theexogenous gene, wherein the signal peptide directs cotranslationalexport of the protein from the cytoplasm to the periplasm. In someembodiments, the signal peptide is derived from the Sec and Tatsecretion pathways. In particular embodiments, the signal peptide isderived from DsbA, pelB, OmpA, TolB, MalE, lpp, TorA, or HylA.

In some embodiments, the culture medium has an OD₆₀₀ between about 0.1to about 500; between about 0.2 to about 100, between about 0.5 to about10, between about 1 to about 2. In exemplary embodiments, the medium hasan OD₆₀₀ of about 1.1.

Expression of the exogenous gene may be induced for about 1 hour toabout 1 week; for about 1 hour to about 1 day; for about 1 hour to about10 hours; for about 10 hours to about 1 week; for about 10 hours toabout 1 day; for about 1 day to about 1 week.

The yield of the protein of interest may be about 0.1 g/L medium toabout 500 g/L medium; about 1 g/L medium to about 500 g/L medium; about1 g/L medium to about 100 g/L medium; about 1 g/L medium to about 10 g/Lmedium; about 10 g/L medium to about 500 g/L medium; about 10 g/L mediumto about 100 g/L medium; about 100 g/L medium to about 500 g/L medium.The yield of the protein of interest may be about 10 mg/L medium toabout 10³ mg/L medium; about 20 mg/L medium to about 500 mg/L medium;about 100 mg/L medium to about 250 mg/L medium; or about 1 mg/L mediumto about 10 mg/L medium. The yield of the protein of interest may beincreased by at least about 2-fold, 3-fold, 5-fold, 8-fold, 10-fold, ascompared to a method of making the protein of interest from geneticallyidentical cells in an unswitched state. The yield of the protein ofinterest may be increased by from about 2-fold to about 100-fold, fromabout 2-fold to about 50-fold, from about 5-fold to about 25-fold, orfrom about 10-fold to about 20-fold. The yield of the protein ofinterest may be increased by from about 2-fold to about 10-fold, fromabout 2-fold to about 20-fold, or from about 2-fold to about 30-fold, ascompared to a method of making the protein of interest from geneticallyidentical cells in an unswitched state.

In some embodiments, the increased protein yields are increased ascompared to methods of making a protein of interest from geneticallyidentical cells under conditions that do not induce a switched L-form(e.g., wherein said conditions that do not induce a switched form areotherwise identical to conditions that provide increased protein ofinterest). In some cases, the conditions that do not induce a switchedL-form comprise the absence of, or an insufficient amount of, one ormore antibiotics that inhibit peptidoglycan biogenesis in the cell. Insome cases, the conditions that do not induce a switched formadditionally or alternatively comprise a magnesium concentration of lessthan 6 mM, less than 5 mM, less than 4 mM, less than 3 mM, less than 2mM, or about 1 mM. In some cases, the conditions that do not induce aswitched form additionally or alternatively comprise conditions in whichthe bacterial cell comprises a periplasmic space that is about 5% toabout 30% or about 10% to about 20% of total cell volume.

In another aspect, the present invention provides a fermentation vesselcontaining any one of the foregoing cell cultures, wherein the cellculture contained by the fermentation vessel comprises a volume ofmedium of about, or of at least about, 1 L; 10 L; 100 L; 250 L; 500 L;or 1,000 L. In some cases, the fermentation vessel is a component of afermentation system. In some cases, the fermentation system furthercomprises modules for controlling oxygen, carbon, or pH, or acombination of 2 or 3 thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the physiological state difference between switched andunswitched cells. A) Unswitched Escherichia coli cells. B) SameEscherichia coli population as figure A but has undergone thephysiological switch. C) Phase contrast of switched Escherichia colicell containing cytoplasmic RFP and periplasmic GFP. D) Fluorescentimaging of cell in figure C illustrates targeted protein localization.

FIG. 2 depicts enhanced protein production in switched cells. A-B)Target protein for T7 inducible protein production is periplasmicexpressed GFP, produced in Escherichia coli BL21. The same population ofcells was used and induced at OD 1.1. A) Protein ladder (lane 1), IPTGinduced protein production (lane 2), IPTG induced protein productionwith physiological switch (lane 3). B) Two vials of the cell GFP inducedcultures with IPTG only on left and IPTG+Switch on right. C) Expressionof a 22KD collagen using switched cells showing protein ladder (lane 1),supernatent after protein production (lane 2), cell pellet (lane 3).

FIG. 3 depicts a timelapse of Escherichia coli cell switching over time.

FIG. 4 illustrates other organisms undergoing the physiological switch.A) Agrobacterium tumefaciens normal physiology. B) Agrobacteriumtumefaciens switched physiology. C) Pseudomonas aeruginosa PAO1 normalphysiology. D) Pseudomonas aeruginosa PAO1 switched physiology. E)Brevundimonas diminuta normal physiology. F) Brevundimonas diminutaswitched physiology. G) Agrobacterium tumefaciens normal physiology. H)Agrobacterium tumefaciens switched physiology.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments of thedisclosure. However, one skilled in the art will understand that thedisclosure may be practiced without these details.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present disclosure. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. It is appreciated that certain features of theinvention, which are, for clarity, described in the context of separateembodiments, may also be provided in combination in a single embodiment.Conversely, various features of the invention, which are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any suitable subcombination.

As used herein the term “about” refers to ±10%.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this disclosure maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of thedisclosure. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

L-Form Bacteria

The present invention provides a protein production platform comprisingmodified bacterial cells exhibiting a novel physiological switchphenotype (L-form) which inhibits cell division and promotes the growthof the periplasmic space in comparison to the cytoplasmic space.Recombinant protein production in these cells is dramatically increasedcompared to that in non-switched cells. This has been tested in severalspecies of Gram negative bacteria (Gammaproteobacteria: Escherichiacoli, Vibrio natriegens, and Pseudomonas fluorescens; andAlphaproteobacteria: Caulobacter crescentus, Agrobacterium tumefaciens,and Brevundimonas diminuta), which suggest a conserved mechanism thatcan be applied to all gram-negative recombinant protein production.These cells still contain and inner and outer membrane but lack afunctional peptidoglycan cell wall. The cell shape is spherical andincreases in volume over time. While the periplasmic space normallycomprises only 10-20% of the total cell volume, the periplasmiccompartment of the switched state of the subject invention can compriseup to 90% of total cell volume. Remarkably, and unexpectedly, the cellsremain viable and are able to undergo metabolic processes and producerecombinant proteins of interest.

The term “recombinant bacterial cell” as used herein refers to a cellthat has been engineered to express a target protein.

The term “coccus” as used herein refers to a bacterial cell having aspherical morphology. Generally the longest dimension of a coccus formbacterial cell is no more than 25%, 40%, 50%, or 100% larger than theshortest dimension.

The term “bacillus” as used herein refers to a bacterial cell having arod-shaped morphology. Typically the longest dimension of a bacillusform bacterial cell is greater than twice the length of the shortestdimension. As such, the longest dimension of a bacillus form bacterialcell can be at least 3, 4, 5, 6, 7, 8, 9, or 10 times the length of theshortest dimension.

The term “periplasmic volume” refers to the total volume contained inthe periplasm, which is the region between the outer membrane and theplasma membrane of the bacterial cell.

The term “cytoplasmic volume” refers to the total volume contained inthe cytoplasm, which is the region inside the plasma membrane of thebacterial cell.

Target Proteins

The present invention provides a platform of recombinant bacterial cellscomprising exogenous genes for producing a protein of interest or“target protein” which is heterologous to the host. A protein or nucleicacid (e.g., nucleic acid encoding a protein, expression cassette, orexpression vector) is heterologous to the host if it is not naturallyoccurring in the host, or is present in the host in a non-naturallyoccurring context (e.g., a non-natural genomic location or a non-naturalsubcellular location). For example, a naturally occurring gene can beoperably linked to a promoter that is not operably linked to thenaturally occurring gene in a corresponding wild-type organism, therebyforming a heterologous expression cassette in a modified bacterial cell.As another example, a naturally occurring genomic fragment that does notnaturally exist in a plasmid in a wild-type bacterium can be subclonedinto a plasmid and transformed into that bacterium, thereby forming aheterologous plasmid in a modified bacterial cell. The term “exogenousgene” refers to a gene that is introduced into the host organism by genetransfer. In some embodiments, exogenous genes encoding the targetproteins of the invention are incorporated into expression vectors,which can be extrachromosomal or designed to integrate into the genomeof the host cell into which it is introduced. Expression of theexogenous genes may be constitutive or inducible.

In some embodiments, the exogenous gene (e.g., cDNA or genomic DNA) usedto produce the recombinant protein of interest, is suitably insertedinto a replicable vector for expression in the bacterium under thecontrol of a suitable promoter for bacteria. Many vectors are availablefor this purpose, and selection of the appropriate vector will dependmainly on the size of the nucleic acid to be inserted into the vectorand the particular host cell to be transformed with the vector. Eachvector contains various components depending on its function(amplification of DNA or expression of DNA) and the particular host cellwith which it is compatible. Expression vectors can contain any numberof appropriate regulatory sequences (including, but not limited to,transcriptional and translational control sequences, promoters,ribosomal binding sites, enhancers, origins of replication, etc.) orother components (selection genes, etc.), all of which are operablylinked as is well known in the art.

Expression vectors contain a nucleic acid sequence that enables thevector to replicate in one or more selected host cells. Such sequencesare well known for a variety of bacteria. The origin of replication fromthe plasmid pBR322 is suitable for most Gram-negative bacteria.

Expression vectors also generally contain a selection gene, also termeda selectable marker. This gene encodes a protein necessary for thesurvival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli. One example of a selectionscheme utilizes a drug to arrest growth of a host cell. Those cells thatare successfully transformed with an exogenous gene produce a proteinconferring drug resistance and thus survive the selection regimen. Inone embodiment, the expression vector selection gene is not a gene thatencodes for resistance to an inhibitor of peptidoglycan biogenesis inthe bacterial cell.

The expression vector for producing a target protein may also contain aninducible promoter that is recognized by the host bacterial organism andis operably linked to the nucleic acid encoding the target protein.Inducible promoters suitable for use with bacterial hosts include theβ-lactamase and lactose promoter systems (Chang et al., Nature, 275:615(1978); Goeddel et al., Nature, 281:544 (1979)), the arabinose promotersystem, including the araBAD promoter (Guzman et al., J. Bacterid., 174:7716-7728 (1992); Guzman et al., J. Bacterid., 177:4121-4130 (1995);Siegele and Hu, Proc. Natl. Acad. Sci. USA, 94:8168-8172 (1997)), therhamnose promoter (Haldimann et al., J. Bacterid., 180:1277-1286(1998)), the alkaline phosphatase promoter, a tryptophan (trp) promotersystem (Goeddel, Nucleic Acids Res., 8:4057 (1980) and EP 36,776), theP_(LtetO-1) and P_(lac/ara-1) promoters (Lutz and Bujard, Nucleic AcidsRes., 25:1203-1210 (1997)), and hybrid promoters such as the tacpromoter. deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983).However, other known bacterial inducible promoters andlow-basal-expression promoters are suitable. Their nucleotide sequenceshave been published, thereby enabling a skilled worker operably toligate them to DNA encoding the target protein.

Promoters for use in bacterial systems may also contain a Shine-Dalgarno(SD) sequence operably linked to the DNA encoding the target protein.The promoter can be removed from the bacterial source DNA by restrictionenzyme digestion and inserted into the vector containing the desiredDNA.

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques commonlyknown to those of skill in the art. Isolated plasmids or DNA fragmentsare cleaved, tailored, and re-ligated in the form desired to generatethe plasmids required. Instructions for handling DNA, digestion andligation of DNA, transformation and selection of transformants can befound inter alfa in the known manual by Sambrook et al. “MolecularCloning: A Laboratory Manual, Second Edition (Cold Spring HarborLaboratory Press, 1989).

The term “gene” as used herein refers to a nucleic acid fragment thatexpresses a specific protein, and which may refer to the coding regionalone or may include regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.

The term “expression vector” refers to an assembly which is capable ofdirecting the expression of the exogenous gene. The expression vectormay include a promoter which is operably linked to the exogenous gene.

The term “constitutive” as used herein refers to an exogenous gene thatis expressed and not known to be subject to regulation that completelycauses cessation of expression under most environmental anddevelopmental conditions.

The term “inducible” as used herein refers to an exogenous gene that isexpressed in response to presence of an inducer such as an exogenouschemical, heat, or light. Standard procedures may be followed to induceprotein production in the L-form bacterial cells described herein. Insome embodiments, the strain BL21(DE3) containing the plasmid pET28a maybe used to drive the IPTG/lactose inducible production of recombinantproteins.

The term “target protein” as used herein refers generally to peptidesand proteins having more than about 10 amino acids. The target proteinsare preferably mammalian proteins. Target proteins generally excludefluourescent proteins. In some embodiments the target protein is otherthan mCherry. In some embodiments the target protein is other than greenfluorescent protein (GFP).

The term “therapeutic protein” as used herein refers to those proteinsthat have demonstrated biological activity and may be employed to treata disease or disorder by delivery to a patient in need thereof by anacceptable route of administration. The biological activity oftherapeutic proteins may be demonstrated in vitro or in vivo and resultsfrom interaction of the protein with receptors and/or otherintracellular or extracellular components leading to a biologicaleffect. Example of therapeutic proteins include, but are not limited to,molecules such as, e.g., renin, a growth hormone, including human growthhormone; bovine growth hormone; growth hormone releasing factor;parathyroid hormone; thyroid stimulating hormone; lipoproteins;al-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;thrombopoietin; follicle stimulating hormone; calcitonin; luteinizinghormone; glucagon; clotting factors such as factor VIIIC, factor IX,tissue factor, and von Willebrands factor; anti-clotting factors such asProtein C; atrial naturietic factor; lung surfactant; a plasminogenactivator, such as urokinase or human urine or tissue-type plasminogenactivator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumornecrosis factor-alpha; tumor necrosis factor-beta; enkephalinase; aserum albumin such as human serum albumin; mullerian-inhibitingsubstance; relaxin A-chain; relaxin B-chain; prorelaxin; mousegonadotropin-associated peptide; a microbial protein, such asbeta-lactamase; DNase; inhibin; activin; vascular endothelial growthfactor (VEGF); receptors for hormones or growth factors; integrin;protein A or D; rheumatoid factors; a neurotrophic factor such asbrain-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6(NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-β;cardiotrophins (cardiac hypertrophy factor) such as cardiotrophin-1(CT-1); platelet-derived growth factor (PDGF); fibroblast growth factorsuch as aFGF and bFGF; epidermal growth factor (EGF); transforminggrowth factor (TGF) such as TGF-alpha and TGF-beta, including TGF-β1,TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factor-I and -II(IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growthfactor binding proteins; CD proteins such as CD-3, CD-4, CD-8, andCD-19; erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-13; anti-HER-2antibody; superoxide dismutase; T-cell receptors; surface membraneproteins; decay accelerating factor; viral antigen such as, for example,a portion of the AIDS envelope; transport proteins; homing receptors;addressins; and regulatory proteins.

In some embodiments the target protein is an antibody. Antibodiesproduced by the claimed invention may be monoclonal antibodies that arehomogeneous populations of antibodies to a particular antigenicdeterminant (e g., a cancer cell antigen, a viral antigen, a microbialantigen, a protein, a peptide, a carbohydrate, a chemical, nucleic acid,or fragments thereof). Such antibodies may be of any immunoglobulinclass including IgG, IgM, IgE, IgA, and IgD and any subclass thereof.

Useful monoclonal antibodies include, but are not limited to, humanmonoclonal antibodies, humanized monoclonal antibodies, antibodyfragments, or chimeric human-mouse (or other species) monoclonalantibodies.

The antibody can also be a bispecific antibody. Bispecific antibodiesmay have a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm.This asymmetric structure facilitates the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation (WO94/04690; Suresh et al. (1986) Methods in Enzymology, 121:210; Rodrigueset al. (1993) J. of Immunology 151:6954-6961; Carter et al. (1992)Bio/Technology 10:163-167; Carter et al. (1995) J. of Hematotherapy4:463-470; Merchant et al. (1998) Nature Biotechnology 16:677-681.

The antibody, as defined, can be a functionally active fragment,derivative or analog of an antibody that immunospecifically binds tocancer cell antigens, viral antigens, or microbial antigens or otherantibodies bound to tumor cells or matrix. In this regard, “functionallyactive” means that the fragment, derivative or analog is able to elicitanti-anti-idiotype antibodies that recognize the same antigen that theantibody from which the fragment, derivative or analog is derivedrecognized. Specifically, in an exemplary embodiment the antigenicity ofthe idiotype of the immunoglobulin molecule can be enhanced by deletionof framework and CDR sequences that are C-terminal to the CDR sequencethat specifically recognizes the antigen. To determine which CDRsequences bind the antigen, synthetic peptides containing the CDRsequences can be used in binding assays with the antigen by any bindingassay method known in the art, e.g. the BIA core assay (Kabat et al.(1991) in Sequences of Proteins of Immunological Interest, FifthEdition, National Institute of Health, Bethesda, Md.; Kabat et al.(1980) J. of Immunology 125(3):961-969).

Other useful antibodies include fragments of antibodies such as, but notlimited to, F(ab′)2 fragments, which contain the variable region, thelight chain constant region and the CH1 domain of the heavy chain, canbe produced by pepsin digestion of the antibody molecule, and Fabfragments, which can be generated by reducing the disulfide bridges ofthe F(ab′)2 fragments. Other useful antibodies are heavy chain and lightchain dimers of antibodies, or any minimal fragment thereof such as Fvsor single chain antibodies (SCAs) (e.g., as described in U.S. Pat. No.4,946,778; Bird (1988) Science 242:423-42; Huston et al., (1988) Proc.Natl. Acad. Sci. U.S.A. 85:5879-5883; and Ward et al. (1989) Nature334:544-54), or any other molecule with the same specificity as theantibody.

The antibody may be a fusion protein of an antibody, or a functionallyactive fragment thereof, for example in which the antibody is fused viaa covalent bond (e.g., a peptide bond), at either the N-terminus or theC-terminus to an amino acid sequence of another protein (or portionthereof, such as at least 10, 20 or 50 amino acid portion of theprotein) that is not the antibody. The antibody or fragment thereof maybe covalently linked to the other protein at the N-terminus of theconstant domain.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal. (1984) Proc. Natl. Acad. Sci. U.S.A., 81:6851-6855). A chimericantibody is a molecule in which different portions are derived fromdifferent animal species, such as those having a variable region derivedfrom murine monoclonal and human immunoglobulin constant regions (U.S.Pat. Nos. 4,816,567; 4,816,397). Chimeric antibodies include“primatized” antibodies comprising variable domain antigen-bindingsequences derived from a non-human primate (e.g., Old World Monkey, Apeetc.) and human constant region sequences.

Therapeutic monoclonal antibodies that may be produced by the bacteriaand methods of the invention include, but are not limited to,trastuzumab (HERCEPTIN®, Genentech, Inc., Carter et al. (1992) Proc.Natl. Acad. Sci. U.S.A., 89:4285-4289; U.S. Pat. No. 5,725,856);anti-CD20 antibodies such as chimeric anti-CD20 “C2B8” (U.S. Pat. No.5,736,137); rituximab (RITUXAN®), ocrelizumab, a chimeric or humanizedvariant of the 2H7 antibody (U.S. Pat. No. 5,721,108; WO 04/056312) ortositumomab (BEXXAR®); anti-IL-8 (St John et al. (1993) Chest, 103:932,and WO 95/23865); antibodies targeting other interleukins, such as IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, and IL-13;anti-VEGF antibodies including humanized and/or affinity maturedanti-VEGF antibodies such as the humanized anti-VEGF antibody huA4.6.1bevacizumab (AVASTIN®, Genentech, Inc., Kim et al. (1992) Growth Factors7:53-64; WO 96/30046; WO 98/45331); anti-PSCA antibodies (WO 01/40309);anti-CD40 antibodies, including S2C6 and humanized variants thereof (WO00/75348); anti-CD11a (U.S. Pat. No. 5,622,700; WO 98/23761; Steppe etal. (1991) Transplant Intl. 4:3-7; Hourmant et al. (1994)Transplantation 58:377-380); anti-IgE (Presta et al. (1993) J. Immunol.151:2623-2632; WO 95/19181); anti-CD18 (U.S. Pat. No. 5,622,700; WO97/26912); anti-IgE, including E25, E26, and E27 (U.S. Pat. Nos.5,714,338 and 5,091,313; WO 93/04173; U.S. Pat. No. 5,714,338);anti-Apo-2 receptor antibody (WO 98/51793); anti-TNF-alpha antibodiesincluding cA2 (REMICADE®), CDP571, and MAK-195 (U.S. Pat. No. 5,672,347;Lorenz et al. (1996) J. Immunol. 156(4): 1646-1653; Dhainaut et al.(1995) Crit. Care Med. 23(9):1461-1469); anti-Tissue Factor (TF) (EP 0420 937 B1); anti-human alpha-4 beta 7 integrin (WO 98/06248);anti-EGFR, chimerized or humanized 225 antibody (WO 96/40210); anti-CD3antibodies such as OKT3 (U.S. Pat. No. 4,515,893); anti-CD25 or anti-tacantibodies such as CHI-621 SIMULECT® and ZENAPAX® (U.S. Pat. No.5,693,762); anti-CD4 antibodies such as the cM-7412 antibody (Choy etal. (1996) Arthritis Rheum 39(1):52-56); anti-CD52 antibodies such asCAMPATH-1H (Riechmann et al. (1988) Nature 332: 323-337); anti-Fcreceptor antibodies such as the M22 antibody directed against Fc gammaRI as in Graziano et al. (1995) J. Immunol. 155(10):4996-5002;anti-carcinoembryonic antigen (CEA) antibodies such as hMN-14 (Sharkeyet al. (1995) Cancer Res. 55(23 Suppl): 5935s-5945s; antibodies directedagainst breast epithelial cells including huBrE-3, hu-Mc 3 and CHL6(Ceriani et al. (1995) Cancer Res. 55(23):5852s-5856s; and Richman etal. (1995) Cancer Res. 55(23 Supp):5916s-5920s); antibodies that bind tocolon carcinoma cells such as C242 (Litton et al. (1996) Eur J. Immunol.26(1):1-9); anti-CD38 antibodies, e.g., AT 13/5 (Ellis et al. (1995) J.Immunol. 155(2):925-937); anti-CD33 antibodies such as Hu M195 (Jurcicet al. (1995) Cancer Res 55(23 Suppl):5908s-5910s) and CMA-676 orCDP771; anti-CD22 antibodies such as LL2 or LymphoCide (Juweid et al.(1995) Cancer Res 55(23 Suppl):5899s-5907s); anti-EpCAM antibodies suchas 17-1A (PANOREX®); anti-GpIIb/IIIa antibodies such as abciximab orc7E3 Fab (REOPRO®); anti-RSV antibodies such as MEDI-493 (SYNAGIS®);anti-CMV antibodies such as PROTOVIR®); anti-HIV antibodies such asPRO542; anti-hepatitis antibodies such as the anti-Hep B antibodyOSTAVIR®); anti-CA 125 antibody OvaRex; anti-idiotypic GD3 epitopeantibody BEC2; anti-human renal cell carcinoma antibody such as ch-G250;ING-1; anti-human 17-1A antibody (3622W94); anti-human colorectal tumorantibody (A33); anti-human melanoma antibody R24 directed against GD3ganglioside; anti-human squamous-cell carcinoma (SF-25); and anti-humanleukocyte antigen (HLA) antibodies such as Smart ID10 and the anti-HLADR antibody Oncolym (Lym-1).

In some embodiments the target protein is non-therapeutic protein.Non-therapeutic proteins include, but are not limited to collagen andderivatives thereof, albumin, ovalbumin, rennet, fibrin, casein(including αS1, αS2, β, κ), elastin, keratin, myosin, fibronectin,laminin, nidogen-1, vitronectin, silk fibroins, prolyl hydroxylases,lysyl hydroxylases, glycosyltransferases, hemeproteins and any otherstructural proteins and enzymes of commercial and/or academic interest.

In some embodiments the target protein is collagen. The term “collagen”as used herein refers to the main protein of connective tissue that hasa high tensile strength and that has been found in most multicellularorganisms. Collagen is a major fibrous protein, and it is also thenonfibrillar protein in basement membranes. It contains an abundance ofglycine, proline, hydroxyproline, and hydroxylysine. Currently, collagentypes I-XIX have been identified and they differ by the amino acidstructure of the alpha chain. The term “collagen” as used herein isunderstood as meaning all collagen types and any form of collagen,whether native nor not, atelocollagen, insoluble collagen, collagenfibers, soluble collagen, and acid-soluble collagen.

Growth Media

Growth media suitable for culturing the L-form bacteria described hereincomprise at least 4 mM magnesium, preferably greater than 4 mM, morepreferably greater than 5 mM, and still more preferably greater than 6mM magnesium concentration, which may be in the form of either magnesiumsulfate (MgSO₄), magnesium chloride (MgCl₂), or other magnesium saltsknown in the art. The media may also contain an osmotic stabilizer suchas sucrose, glucose, or a betaine. In some embodiments, theconcentration of osmotic stabilizer should be at least about 3%, 4% or5% weight/volume.

The term “osmotic stabilizer” as used herein refers to a component usedto control the osmotic strength of the medium and reduce turgor pressureinside the bacterial cells. Osmotic stabilizers can include, but are notlimited to, e.g. sugars (e.g., arabinose, glucose, sucrose, glycerol,sorbitol, mannitol, fructose, galactose, saccharose,maltotrioseerythritol, ribitol, pentaerythritol, arabitol, galactitol,xylitol, iditol, maltotriose, and the like), betaines (e.g.,trimethylglycine), proline, one or more salts such as an ammonium,potassium, or sodium salt (e.g., sodium chloride), one or more polymers(e.g., polyethylene glycol, polyethylene glycol monomethylether,polysucrose, polyvinylpyrrolidone, polypropylene glycol), or acombination thereof. In some cases, the concentration of the osmoticstabilizer(s) in the medium is at least about 4%, 5%, 6%, or 7% (w/v).In further embodiments, the concentration of osmotic stabilizer is atleast about 8%, 9%, or 10% (w/v). In some embodiments, the concentrationof the osmotic stabilizer in the medium is between about 5% to about 20%(w/v).

In some embodiments, the total concentration of osmotic stabilizer(e.g., one or more of the osmotic stabilizers described above) issufficient to provide a culture medium having an osmolality equal to theosmolality of switch media 1, switch media 2, or bioreactor media MGZ12,described hereinbelow in Example 1. In some embodiments, the the totalconcentration of osmotic stabilizer (e.g., one or more of the osmoticstabilizers described above) is sufficient to provide a culture mediumhaving an osmolality that is from about 50% lower to about 50% higherthan the osmolality of switch media 1, switch media 2, or bioreactormedia MGZ12, described hereinbelow in Example 1. In some embodiments,the the total concentration of osmotic stabilizer (e.g., one or more ofthe osmotic stabilizers described above) is sufficient to provide aculture medium having an osmolality that is from about 25% lower toabout 25% higher, or from about 10% lower to about 10% higher than theosmolality of switch media 1, switch media 2, or bioreactor media MGZ12,described hereinbelow in Example 1.

The term “sugar” as used herein refers to reducing sugars (e.g.,cellobiose, fructose, galactose, glucose, glyceraldehyde, lactose,maltose, and ribose), non-reducing sugars (e.g., melezitose, melibiose,raffinose, sorbose, sucralose, sucrose, trehalose, and verbascose), andsugar alcohols (e.g., amltitol, arabitol, dulcitol, erythritol,glycerol, glycol, iditol, isomalt, lactitol, mannitol, rebitol,sorbitol, threitol, and xylitol)

The term “betaine” as used herein refers to fully N-methylated aminoacids, including, but not limited to trimethylglycine.

Salts and other nutrients should be added to the media to supplementgrowth. Salts and media compositions that support the physiologicalswitch physiology that have been tested are M63 salt media, M9 saltmedia, PYE media, and Luria-Bertani (LB) media. Any necessarysupplements besides carbon, nitrogen, and inorganic phosphate sourcesmay also be included at appropriate concentrations introduced alone oras a mixture with another supplement or medium such as a complexnitrogen source. In certain embodiments, the medium further comprisesone or more ingredients selected from: ammonium chloride, ammoniumsulfate, calcium chloride, casamino acids, iron(II) sulfate, magnesiumsulfate, peptone, potassium phosphate, sodium chloride, sodiumphosphate, and yeast extract.

In some embodiments, the cell culture is free of animal-derivedcomponents. In some embodiments, the cell culture comprises a definedmedium. In some embodiments, the cell culture is free of yeast extract.

In some embodiments, the cell culture comprises from about 1×108bacterial cells per mL of culture volume to about 1×1010 bacterial cellsper mL in a volume of at least about 1 L (e.g., from about 1 L to about500,000 L, from about 1 L to about 10,000 L, from about 1 L to about1,000 L, from about about 1 L to about 500 L, or from about 1 L to about250 L). In some embodiments, the cell culture comprises from about 4×108bacterial cells per mL of culture volume to about 1×109 bacterial cellsper mL in a volume of at least about 1 L (e.g., from about 1 L to about500,000 L, from about 1 L to about 10,000 L, from about 1 L to about1,000 L, from about about 1 L to about 500 L, or from about 1 L to about250 L).

In certain embodiments, the medium also contains a selection agent,chosen based on the construction of the expression vector, toselectively permit growth of prokaryotic cells containing the expressionvector. For example, kanamycin is added to media for growth of cellsexpressing a kanamycin resistant gene.

In some embodiments, the cell culture comprises at least one exogenousantibiotic inhibitor of bacterial cell peptidoglycan biogenesis. Anexogenous antibiotic inhibitor is an antibiotic as is commonlyunderstood in the art and includes antimicrobial agents naturallyproduced by microorganisms such as bacteria (including Bacillusspecies), actinomycetes (including Streptomyces) or fungi that inhibitgrowth of or destroy other microbes, or genetically-engineered thereofand isolated from such natural source. Substances of similar structureand mode of action can be synthesized chemically, or natural compoundscan be modified to produce semi-synthetic antibiotics. Exemplary classesof antibiotics include, but are not limited to, (1) β-lactams, includingthe penicillins, cephalosporins monobactams, methicillin, andcarbapenems; (2) aminoglycosides, e.g., gentamicin, kanamycin, neomycin,tobramycin, netilmycin, paromomycin, and amikacin; (3) tetracyclines,e.g., doxycycline, minocycline, oxytetracycline, tetracycline, anddemeclocycline; (4) sulfonamides (e.g., mafenide, sulfacetamide,sulfadiazine and sulfasalazine) and trimethoprim; (5) quinolones, e.g.,ciprofloxacin, norfloxacin, and ofloxacin; (6) glycopeptides (e.g.,vancomycin, telavancin, teicoplanin); (7) macrolides, which include forexample, erythromycin, azithromycin, and clarithromycin; (8) carbapenems(e.g., ertapenem, doripenem, meropenem, and imipenem); (9)cephalosporins (e.g., cefadroxil, cefepime, and ceftobiprole); (10)lincosamides (e.g., clindamycin, and lincomycin); (11) monobactams(e.g., aztreonam); (12) nitrofurans (e.g., furazolidone, andnitrofurantoin); (13) Penicillins (e.g., amoxicillin, and Penicillin G);(14) polypeptides (e.g., bacitracin, colistin, and polymyxin B); and(15) other antibiotics, e.g., ansamycins, polymycins, carbacephem,chloramphenicol, lipopeptide, and drugs against mycobacteria (e.g., theones causing diseases in mammals, including tuberculosis (Mycobacteriumtuberculosis) and leprosy (Mycobacterium leprae), and any combinationsthereof. An exogenous antibiotic inhibitor of bacterial cellpeptidoglycan biogenesis can be used, present in, or added to a culture,in an amount or concentration effective to inhibit or block bacterialcell peptidoglycan biogenesis. An exogenous antibiotic inhibitor ofbacterial cell peptidoglycan biogenesis can be used, present in, oradded to a culture, in an amount effective to inhibit or block bacterialcell division. An exogenous antibiotic inhibitor of bacterial cellpeptidoglycan biogenesis can be used, present in, or added to a culture,in an amount or concentration effective to kill a bacterial cell in aculture medium containing less than 6, 5, 4, 3, or about 1 mM magnesiumand/or osmotic stabilizers. An exogenous antibiotic inhibitor ofbacterial cell peptidoglycan biogenesis can be used, present in, oradded to a culture, in an amount or concentration that is at or above aminimum inhibitory concentration of the antibiotic inhibitor in controlmedium that does not induce a switched form. An exogenous antibioticinhibitor of bacterial cell peptidoglycan biogenesis can be used,present in, or added to a culture, in an amount or concentration that iseffective to reduce colony formation of bacteria on a test plate (e.g.,LB agar) by at least about 95% or 99% as compared to the absence of theexogenous antibiotic inhibitor in a control test plate.

In some embodiments, the cell culture comprises at least twostructurally distinct exogenous antibiotic inhibitors of bacterial cellpeptidoglycan biogenesis. In some cases, the at least two structurallydistinct exogenous antibiotic inhibitors of bacterial cell peptidoglycanbiogenesis inhibit different components of a peptidoglycan biogenesispathway in the bacterial cell (e.g., the at least two structurallydistinct exogenous antibiotic inhibitors inhibit different enzymes ofthe peptidoglycan biogenesis pathway in the bacterial cell). In somecases, the cell culture comprises an exogenous antibiotic inhibitor of atransglycosylase component of bacterial cell peptidoglycan biogenesis.In some cases, the cell culture comprises an exogenous antibioticinhibitor of a transpeptidase component of bacterial cell peptidoglycanbiogenesis. In some cases, the cell culture comprises an exogenousantibiotic inhibitor of UDP-N-acetylmuramyl (UDP-MurNAc)-pentapeptidebiogenesis or UDP-N-acetylglucosamine (UDP-GlcNAc) biogenesis.

In some cases, the cell culture comprises an exogenous antibioticinhibitor of MurA, MurB, MurC, MurD, MurE, MurF, MraY, MurG, FemX, FemA,FemB, FtsW, or a penicillin binding protein (PBP). In some cases, thecell culture comprises at least two structurally distinct exogenousantibiotic inhibitors, wherein the at least two structurally distinctexogenous antibiotic inhibitors inhibit different proteins selected fromMurA, MurB, MurC, MurD, MurE, MurF, MraY, MurG, FemX, FemA, FemB, FtsW,or a penicillin binding protein (PBP). In some embodiments, the cellculture comprises at least one or at least two structurally distinctantibiotic(s) selected from: β-lactam antibiotics, phosphonic acidantibiotics, polypeptide antibiotics, D-cycloserine, and glycopeptideantibiotics, wherein the antibiotic(s) inhibit peptidoglycan biogenesisin the bacterial cell.

In some embodiments, the culture medium comprises at least one reactiveoxygen species (ROS) scavenger (e.g., reduced glutathione (GSH), or athiol containing non-peptidic small molecule having a molecular weightfrom about 70 g/mol to about 350 g/mol, or from about 75 g/mol to about155 g/mol).

In some embodiments, the culture medium comprises an antibiotic (e.g.,second or third antibiotic) that selects for the presence of aselectable marker in an expression vector that comprises an exogenousgene encoding for a protein of interest. In some embodiments, theantibiotic that selects for the presence of a selectable marker in theexpression vector is not an inhibitor of peptidoglycan biogenesis in thebacterial cell.

Physiological Switch

Without being bound by theory, the cell morphology that promotesrecombinant protein production and inhibits cell division appears to bedriven by the removal of the cell wall under the media conditions statedabove. In some embodiments, the methods for removal/inhibition of cellwall synthesis can be through the use of antibiotics that inhibitpeptidoglycan synthesis (such as ampicillin, carbenicillin, penicillinsor fosfomycin), or other methods known in the art.

In some embodiments, the antibiotic is selected from: β-lactamantibiotics, phosphonic acid antibiotics, polypeptide antibiotics, andglycopeptide antibiotics. In some embodiments, the antibiotic is anantibiotic selected from: penicllins, cephalosporins, carbapenems, andmonobactams. In some embodiments, the antibiotic is selected from:alafosfalin, amoxicillin, ampicillin, aztreonam, bacitracin,carbenicillin, cefamandole, cefotaxime, cefsulodin, cephalothin,fosmidomycin, methicillin, nafcillin, oxacillin, penicillin g,penicillin v, fosfomycin, primaxin, D-cycloserine, and vancomycin.

Periplasmic Targeting

When having an appropriate signal sequence, recombinantly producedpolypeptides can be secreted into the periplasmic space of bacterialcells. Joly, J. C. and Laird, M. W., in The Periplasm ed. Ehrmann, M.,ASM Press, Washington D.C., (2007) 345-360. In the chemically oxidizingenvironment of the periplasm the formation of disulfide bonds andthereby the functionally correct folding of polypeptides is favored.

In general, the signal sequence may be a component of the expressionvector, or it may be a part of the exogenous gene that is inserted intothe vector. The signal sequence selected should be one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. For bacterial host cells that do not recognize and processthe native signal sequence of the exogenous gene, the signal sequence issubstituted by any commonly known bacterial signal sequence.

In some embodiments, recombinantly produced polypeptides can be targetedto the periplasmic space using the DsbA signal sequence. Dinh andBernhardt, J Bacteriol, September 2011, 4984-4987. In some embodiments,recombinantly produced polypeptides can be targeted to the periplasmicspace using an DsbA, pelB, OmpA, TolB, MalE, lpp, TorA, or HylA signalsequence. In some embodiments, recombinantly produced polypeptides canbe targeted to the periplasmic space using a portion (at least 10%, 25%,50%, 75%, 95%, 99%) of a gene encoding a protein that is secreted intothe periplasmic space. For example, in some embodiments, recombinantlyproduced polypeptides can be targeted to the periplasmic space using aportion (at least 10%, 25%, 50%, 75%, 95%, 99%) of a gene encoding aprotein DsbA, pelB, OmpA, TolB, MalE, lpp, TorA, or HylA, wherein theportion contains the signal sequence of DsbA, pelB, OmpA, TolB, MalE,lpp, TorA, or HylA respectively. In some embodiments, a recombinantlyproduced polypeptide can be targeted to the periplasmic space by fusingthe gene encoding the polypeptide to a nucleic acid encoding all orsubstantially all of a protein that is secreted into the periplasmicspace, e.g., a protein selected from DsbA, pelB, OmpA, TolB, MalE, lpp,TorA, or HylA, thereby producing a fusion protein. Generally, suchperiplasmic targeting employs an N-terminal fusion in which the signalsequence or portion containing the signal sequence is N-terminal to thepolypeptide of interest.

Fermentative Protein Production

The present invention furthermore provides a process for fermentativepreparation of a protein, comprising the steps of:

-   -   a) culturing a recombinant Gram-negative bacterial cell in a        medium comprising a magnesium salt, wherein the concentration of        magnesium ions in the medium is at least about 6 mM, and wherein        the bacterial cell comprises an exogenous gene encoding the        protein, provided that the protein is other than mCherry or        green fluorescent protein;    -   b) inhibiting peptidoglycan biogenesis in the bacterial cell        (e.g., by adding to the medium 1, 2, or more antibiotics that        inhibit peptidoglycan biogenesis); and    -   c) harvesting the protein from the medium.

The bacteria may be cultured continuously—as described, for example, inWO 05/021772—or discontinuously in a batch process (batch cultivation)or in a fed-batch or repeated fed-batch process for the purpose ofproducing the target protein. In some embodiments, protein production isconducted on a large-scale. Various large-scale fermentation proceduresare available for production of recombinant proteins. Large-scalefermentations have at least 1,000 liters of capacity, preferably about1,000 to 100,000 liters of capacity. These fermentors use agitatorimpellers to distribute oxygen and nutrients, especially glucose (thepreferred carbon/energy source). Small-scale fermentation refersgenerally to fermentation in a fermentor that is no more thanapproximately 20 liters in volumetric capacity.

For accumulation of the target protein, the host cell is cultured underconditions sufficient for accumulation of the target protein. Suchconditions include, e.g., temperature, nutrient, and cell-densityconditions that permit protein expression and accumulation by the cell.Moreover, such conditions are those under which the cell can performbasic cellular functions of transcription, translation, and passage ofproteins from one cellular compartment to another for the secretedproteins, as are known to those skilled in the art.

The bacterial cells are cultured at suitable temperatures. For E. coligrowth, for example, the typical temperature ranges from about 20° C. toabout 39° C. In one embodiment, the temperature is from about 25° C. toabout 37° C. In another embodiment, the temperature is at about 30° C.

The pH of the culture medium may be any pH from about 5-9, dependingmainly on the host organism. For E. coli, the pH is from about 6.8 toabout 7.4, or about 7.0.

For induction, typically the cells are cultured until a certain opticaldensity is achieved, e.g., an OD₆₀₀ of about 1.1, at which pointinduction is initiated (e.g., by addition of an inducer, by depletion ofa repressor, suppressor, or medium component, etc.) to induce expressionof the exogenous gene encoding the target protein.

After product accumulation, the cells can be vigorously stirred or mixed(e.g., vortexed), and/or centrifuged in order to induce lysis andrelease of recombinant proteins. The majority of the proteins aretypically found in the supernant but any remaining membrane boundproteins can be released using detergants (e.g., a non-ionic detergentsuch as triton X-100).

In a subsequent step, the target protein, as a soluble or insolubleproduct released from the cellular matrix, is recovered in a manner thatminimizes co-recovery of cellular debris with the product. The recoverymay be done by any means, but in one embodiment, can comprise ofhistidine tag purification through a nickel colum. See for example,Purification of Proteins Using Polyhistidine Affinity Tags, MethodsEnzymology. 2000; 326: 245-254.

The target protein captured in the initial recovery step may then befurther purified for example by chromatography. General chromatographicmethods and their use are known to a person skilled in the art. See forexample, Chromatography, 5th edition, Part A: Fundamentals andTechniques, Heftmann, E (ed), Elsevier Science Publishing Company, NewYork, (1992); Advanced Chromatographic and Electromigration Methods inBiosciences, Deyl, Z. (ed.), Elsevier Science BV, Amsterdam, TheNetherlands, (1998); Chromatography Today, Poole, C. F., and Poole, S.K., Elsevier Science Publishing Company, New York, (1991); Scopes,Protein Purification Principles and Practice (1982); Sambrook, J., etal. (ed), Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; orCurrent Protocols in Molecular Biology, Ausubel, F. M., et al. (eds),John Wiley & Sons, Inc., New York. The following procedures areexemplary of suitable purification procedures: fractionation onimmunoaffinity or ion-exchange columns; ethanol precipitation;reversed-phase HPLC; chromatography on silica or on a cation-exchangeresin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfateprecipitation; and gel filtration using, for example, SEPHADEX™ G-75.

The following examples are provided for purposes of illustration, notlimitation.

EXAMPLES Example 1 Construction of the Modified Bacteria

Materials and Methods:

Strains:

Tested Physiological Switch and Protein Production:

-   E. coli BL21(DE3)—From NEB, product #c2527-   E. coli K12 NCM3722—From The Coli Genetic Stock Center, CGSC #12355    Tested Physiological Switch:    Gammaproteobacteria:-   Vibrio natriegens—From ATCC, product #14048-   Pseudomonas fluorescens—From ATCC, product #31948-   Pseudomonas aeruginosa PAO1—From ATCC, product #BAA-47    Alphaproteobacteria:-   Caulobacter crescentus—From ATCC, product #19089-   Agrobacterium tumefaciens/Rhizobium radiobacter—From ATCC, product    #33970-   Brevundimonas diminuta—From ATCC, product #13184    Media Compositions:    1 Liter 5× m63 Salts:-   10 g (NH4)2SO4—From P212121, product #7783-20-2-   68 g KH2PO4—From P212121, product #7778-77-0-   2.5 mg FeSO4.7H2O—From Sigma Aldrich, product #F7002-   Bring volume up to 1 liter with milliQ water-   Adjust to pH 7 with KOH (From P212121, product #1310-58-3)-   Autoclave mixture    1 Liter of 1M MgSO4:-   246.5 g MgSO4 7H2O—From P212121, product #10034-99-8-   Bring volume up to 1 liter with milliQ water-   Autoclave mixture    1 Liter of Switch Media 1:-   133.4 mL 5× m63 Salts-   10 mL 1M MgSO4-   38.6 g Glucose—From P212121, product #50-99-7-   66.6 g Sucrose—From P212121, product #57-50-1-   8.33 g LB mix—From P212121, product #lb-miller-   Bring volume up to 1 liter with milliQ water-   Filter sterilize mixture through a 0.22 μM pore vacuum filter (From    Sigma Aldrich, product #CLS430517)    1 Liter of Switch Media 2:-   133.4 mL 5× m63 Salts-   10 mL 1M MgSO4-   38.6 g Glucose—From P212121, product #50-99-7-   66.6 g Sucrose—From P212121, product #57-50-1-   10 g Yeast Extract—From FisherSci.com, product #J60287A1-   Bring volume up to 1 liter with milliQ water-   Filter sterilize mixture through a 0.22 μM pore vacuum filter (From    Sigma Aldrich, product #CLS430517)    For Bioreactor Growth:-   5 liter of bioreactor media MGZ12:-   1) Autoclave 1 L of Glucose at concentration of 500 g/L in DI water.    From VWR, product #97061-170.-   2) Autoclave 1 L of Sucrose at concentration of 500 g/L in DI water.    From Geneseesci.com, product #62-112.-   3) Autoclave in 3946 mL of DI water:-   20 g (NH4)2HPO4. From VWR, product #97061-932.-   66.5 g KH2PO4. From VWR, product #97062-348.-   22.5 g H3C6H5O7. From VWR, product #BDH9228-2.5KG.-   2.95 g MgSO4.7H2O. From VWR, product #97062-134.-   10 mL Trace Metals (Teknova), 1000×. From Teknova, product #T1001.-   After autoclaving add 400 mL of (1) to (3), 65 mL of 10M NaOH (from    VWR, product #97064-480) to (3), and 666 mL of (2) to (3).-   A feed of 500 g/L of glucose can be used during fermentation run as    needed.-   At induction add:-   50 mL of 1M MgSO4.7H2O to a 5 L bioreactor-   1 to 10 mM concentration of IPTG. From carbosynth.com, product    #EI05931-   Add Fosfomycin (50 μg/mL or higher) and Carbenicillin (100 μg/mL or    higher).    Physiological Switch:-   The physiological switch is optimally flipped at an OD 600 of 1 to    1.1 for E. coli for growth in shake flasks at volumes up to 1 L. For    the other species tested, cultures were grown in switch media and    subcultured once cultures reached maximal OD 600. In all cases the    physiological switch is flipped through the addition of 100-200    ug/mL Carbenicillin (From P212121, product #4800-94-6) and 50-100    ug/mL Fosfomycin (From P212121, product #26016-99-9). The majority    of the population is in the switched state within a few hours. To    confirm that cells underwent a physiological switch, cells were    imaged on a Nikon Ti-E with perfect focus system, Nikon CFI60 Plan    Apo 100× NA 1.45 objective, Prior automated filter wheels and stage,    LED-CFP/YFP/mCherry and LED-DA/FI/TX filter sets (Semrock), a    Lumencor Sola II SE LED illumination system, and a Hamamatsu Flash    4.0 V2 CMOS camera.    Image Analysis of Physiological Switch:-   Images were analyzed using ImageJ to measure dimensions. In the    switched state, the spherical outline of the outer membrane is    treated as a sphere to calculate total volume (V=(4/3)πr³). The    cytoplasmic volume is calculated as an ellipsoid that exists within    the sphere (V=(4/3)π*(longest radius)*(short radius)²). To calculate    the periplasmic volume, the cytoplasmic volume is subtracted from    the total volume of the cell.    Protein Expression and Quantification:-   E. coli BL21(DE3) (NEB product #c2527) containing pET28a (emd    Millipore product #69864) and its derivatives carrying GFP or    collagen derivatives were grown in a shaking incubator at 37° C.    overnight in switch media containing 50 mg/mL kanamycin (p212121    product #2251180). Next day, subcultures are started with a 1:10    dilution of the overnight culture into fresh switch media containing    50 mg/mL kanamycin. The culture is then physiologically switched and    protein production is induced simultaneously at an OD 600 of 1 to    1.1 (Read on a Molecular Devices Spectramax M2 microplate reader).    The physiologically switch and protein production are flipped    through the addition of 100 ug/mL Carbenicillin, 50 ug/mL    Fosfomycin, and 100 ug/mL IPTG (p212121 product #367-93-1). Protein    expression is continued in the switched state from between 8 hours    to overnight at room temperature (approximately 22° C.) on an    orbital shaker. In order to quantify total protein levels, Quick    Start™ Bradford Protein Assay was used on mixed portion of culture    and standard curves are quantitated on a Molecular Devices    Spectramax M2 microplate reader. In order to quantitate the relative    intensity of target protein production relative to the rest of the    protein population the mixed portion of the cultures were run on    Mini-PROTEAN® TGX™ Gels and stained with Bio-Safe™ Coomassie Stain.

Induction of Protein Production:

Standard procedures have been followed to induce protein production inthe physiological state. We have been using the strain BL21(DE3)containing the plasmid pET28a driving the IPTG/lactose inducibleproduction of recombinant proteins and targeting them to the periplasmicspace using the DsbA signal sequence. Using the GFP protein, targeted tothe periplasmic space as described above, we have demonstrated theability to gain and increase of 5-fold in protein production whencompared to un-switched cell populations induced at the same opticaldensity, for the same amount of time (figures). The induction wasoptimal at an OD600 of 1.1 and induction was continued for 10 hours atwhich point the protein produced was measured at about 200 mg/mL.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification areincorporated herein by reference, in their entirety to the extent notinconsistent with the present description.

From the foregoing it will be appreciated that, although specificembodiments described herein have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope described herein. Accordingly, the disclosure isnot limited except as by the appended claims.

What is claimed is:
 1. A method of producing collagen comprising: a)culturing a recombinant Gram-negative bacterial cell in a mediumcomprising a magnesium salt, wherein the concentration of magnesium ionsin the medium is at least about 6 mM, and wherein the recombinantGram-negative bacterial cell comprises an exogenous gene encoding thecollagen; b) inhibiting peptidoglycan biogenesis in the recombinantGram-negative bacterial cell; and c) harvesting the collagen from themedium, wherein the inhibiting of b) comprises adding an antibiotic tothe medium, wherein the antibiotic inhibits peptidoglycan biogenesis inthe recombinant Gram-negative bacterial cell.
 2. The method of claim 1,wherein the recombinant Gram-negative bacterial cell is selected fromthe group consisting of: gammaproteobacteria and alphaproteobacteria. 3.The method of claim 2, wherein the recombinant Gram-negative bacterialcell is selected from the group consisting of: Escherichia coli, Vibrionatriegens, Pseudomonas fluorescens, Caulobacter crescentus,Agrobacterium tumefaciens, and Brevundimonas diminuta.
 4. The method ofclaim 2, wherein the recombinant Gram-negative bacterial cell isEscherichia coli.
 5. The method of claim 1, wherein the medium furthercomprises an osmotic stabilizer.
 6. The method of claim 1, wherein therecombinant Gram-negative bacterial cell comprises an expression vectorcomprising the exogenous gene.
 7. The method of claim 1, wherein themethod further comprises inducing expression of the exogenous gene. 8.The method of claim 1, wherein the recombinant Gram-negative bacterialcell further comprises a nucleic acid sequence encoding a signal peptideoperably linked to the exogenous gene, wherein the signal peptidedirects cotranslational export of the collagen from the cytoplasm to theperiplasm.
 9. The method of claim 8, wherein the signal peptide is aperiplasmic disulfide bond oxidoreductase.
 10. The method of claim 1,wherein the collagen is human collagen.
 11. The method of claim 1,wherein the harvesting of c) comprises clarifying the medium bycentrifugation, filtration, or a combination thereof.
 12. The method ofclaim 1, wherein a yield of the collagen is about 1 g/L medium to about500 g/L medium.
 13. The method of claim 1, wherein the recombinantGram-negative bacterial cell has a periplasm and a cytoplasm, wherein aratio of a periplasmic volume to a cytoplasmic volume is between about0.5:1 and about 10:1.
 14. The method of claim 1, wherein the recombinantGram-negative bacterial cell is a coccus having a longest dimension ofabout 2 μm to about 16 μm.
 15. The method of claim 1, wherein theantibiotic is selected from the group consisting of: a β-lactamantibiotic, a phosphonic acid antibiotic, a polypeptide antibiotic, anda glycopeptide antibiotic.
 16. The method of claim 15, wherein theβ-lactam antibiotic is selected from the group consisting of: apenicillin, a cephalosporin, a carbapenem, and a monobactam.
 17. Themethod of claim 1, wherein the antibiotic is selected from the groupconsisting of: alafosfalin, amoxicillin, ampicillin, aztreonam,bacitracin, carbenicillin, cefamandole, cefotaxime, cefsulodin,cephalothin, fosmidomycin, methicillin, nafcillin, oxacillin, penicilling, penicillin v, fosfomycin, primaxin, and vancomycin.
 18. The method ofclaim 1, wherein the inhibiting of b) comprises adding at least twostructurally distinct antibiotics to the medium, wherein the at leasttwo structurally distinct antibiotics inhibit peptidoglycan biogenesisin the recombinant Gram-negative bacterial cell.
 19. The method of claim18, wherein the at least two structurally distinct antibiotics inhibitdifferent components of a peptidoglycan biogenesis pathway in therecombinant Gram-negative bacterial cell.
 20. The method of claim 1,wherein the recombinant Gram-negative bacterial cell is free of a markerencoding for resistance to an inhibitor of bacterial cell peptidoglycanbiogenesis.
 21. The method of claim 6, wherein the medium comprises anantibiotic that selects for the presence of the expression vector,wherein the antibiotic that selects for the presence of the expressionvector is not an inhibitor of a component of peptidoglycan biogenesis inthe recombinant Gram-negative bacterial cell.
 22. The method of claim 5,wherein the osmotic stabilizer is a sugar, a betaine, or a combinationthereof.
 23. The method of claim 22, wherein the sugar is selected fromthe group consisting of: arabinose, glucose, and sucrose.
 24. The methodof claim 22, wherein the betaine is trimethylglycine.
 25. The method ofclaim 5, wherein a concentration of the osmotic stabilizer in the mediumis between 5% w/v to 20% w/v.